The specific aim of this proposal is to solve the three-dimensional x-ray crystallographic structure of a catalytic "hammerhead" ribozyme RNA. The justification for submission of this proposal to the program in "Ribozyme Antisense Mediated Cleavage of HIV RNA" lies in the demonstration that the hammerhead ribozymes can act as sequence-specific endoribonucleases, hence there appears to be a potential to target their RNA cleavage activity to specific RNAs, including (but not limited to) HIV RNA. We would argue that knowing the three-dimensional structure of a hammerhead ribozyme would dramatically enhance our understanding of their mechanism of substrate recognition and catalysis, which in turn would aid in the design of sequence-specific hammerhead endoribonucleases. We have synthesized, purified and crystallized an RNA/DNA hybrid which represents a ribozyme-inhibitor complex. At this point, our best crystals do not diffract well enough for high resolutions structural studies. Hence our first aim is to systematically vary the sequence parameters in our hammerhead RNAs until we find a combination of sequence and crystallization conditions that yield crystals that diffract to high resolution. Once we have suitable crystals, we will exploit several options for structure determination, including (i) multiple isomorphous replacement with both specifically incorporated iodinated and brominated nucleotides, as well as classical "soak and pray" derivatives, and (ii) if required, phase determination by multiwavelength anomalous dispersion, using the anomalous scattering of Br atoms of specifically incorporated brominated nucleotides.